Thyrotropin (thyroid stimulating hormone, TSH), chorionic gonadotropin (CG), lutropin (luteinizing hormone, LH) and follitropin (follicle stimulating hormone, FSH) are members of a glycoprotein hormone family. These hormones are structurally related heterodimers with a common alpha subunit noncovalently linked to a distinct β-subunit that confers the immunological and biological specificity of each hormone. The functional activity of TSH depends on the correct assembly of the subunits into heterodimers. It is believed that the rate-limiting step in the formation of active TSH is the rate of synthesis of the β-subunit by the pituitary thyrotrope cell.
The molecular weight of mammalian TSH can range from 28 to 30 kilodaltons, with variation being associated with heterogeneity of the oligosaccharide chains. The α-subunit has a molecular weight of approximately 14 kilodaltons and has two oligosaccharide units linked to asparagine residues. Like other glycoprotein hormones, TSH is highly glycosylated with N-linked complex carbohydrates, which account for approximately 21% and 12% of the total weight in its α and β chains respectively (Gesundheit, N., Weintraub, B D, Adv. Exp. Biol., 205, 87 (1986)). Assembly of the α- and β-subunits is necessary for the biological activity of the hormone. The β-subunit has a molecular weight of 15 kilodaltons and has one asparagine linked oligosaccharide unit. There are five disulphide bonds in the α subunit and six in the β subunit. The crystal structure of TSH has not been established yet, but the crystal structure of human CG, which is structurally related, reveals that each subunit contains a central cysteine knot and three loops, two β-hairpin loops on one side of cysteine knot and a long loop in the α subunit which contains a two turn α helix on the other side. TSH has thus been classified as a part of CKGF (Cysteine Knot like Growth Factors) superfamily (Lapthorn, et al., Nature, 369, 455 (1994)).
The carbohydrate structure of glycoproteins plays an important role in hormone assembly, secretion and action. High mannose precursor carbohydrate plays an important role in the α/β-subunit heterodimer by minimizing subunit aggregation and intracellular proteolysis. The complex final carbohydrate appears to be important in determining intrinsic biologic activity as well as the metabolic clearance rate of secreted hormone from the circulation. The carbohydrates of the common α-subunit are most important for biological activity (Sairam, M., Bhargavi, G., Science, 229, 65 (1985)). Interestingly, removal of terminal sialic acid residues of recombinant human TSH increased in vitro bioactivy of hormone (Thotakura, et al., Glycobiology, 4, 525 (1995)). The biological activity of resulting peptide showed a reduced ability to stimulate adenylate cyclase, despite still binding to its receptor with high affinity (Thotakura N, Blithe, D., Glycobiology, 5, 3 (1995)).
Thyrotropin (TSH) is produced and released by anterior pituitary gland and, through its action on the thyroid gland, plays a major role in maintaining circulating levels of thyroid hormones. Released TSH binds with membrane bound TSH receptors (TSH-R) on the cells of the thyroid gland. TSH-R is a seven-transmembrane-spanning protein that uses G-protein coupled signal transduction pathways. TSH binding to TSH-R activates the adenylyl cyclase and phosphotidylinositol systems in cells, leading to increased iodination of thyroglobulin by the thyroid. TSH also promotes thyroid cell growth, leading to the formation of a goiter if the thyroid gland is overstimulated by TSH. The thyroid makes two hormones; triiodothyronine (T3) which contains three iodine atoms, and thyroxine (T4) which contains four, which are influenced by TSH levels. TSH synthesis and release is primarily controlled by the concentrations of thyroid hormones (especially T3) and the amount of thyroid releasing hormone (TRH) released by the hypothalamus. Thyroid hormones control, through negative feedback, the mRNA transcription of TSH, while TRH controls the glycosylation, activation, and release of TSH.
Extensive work has been done characterizing the genes and expressed proteins for the alpha and beta subunits of TSH for various species. The alpha and beta subunits are synthesized from separate mRNAs coded by DNA from genes on separate chromosomes. For example, a single gene coding for the alpha subunit of TSH has been isolated and cloned from numerous species including humans (Fiddes, J, Goodman, H. M., Nature, 281, 351 (1979)), cattle (Erwin et al., Biochemistry, 22, 4856 (1983)), rat (Godine et al., J. Biol. Chem., 257, 8368 (1982)), mouse (Chin et al., Proc. Natl. Acad. Sci. USA, 78, 5329 (1981)), horse (M, O., Headon, D., Biochem. Soc. Trans., 3, 347S (1995)), and dog (Yang et al., Domestic Animal Endocrinology, 18, 379 (2000)). This work has shown that there are two N-linked oligosaccharide chains attached to Asn56 and Asn82 and five intramolecular disulphide bonds in the α-subunit. A 24 amino acid leader sequence, which is cleared prior to secretion, is followed by a 96 amino acid mature protein in all species except the man, where the TSH α-subunit is a 92 amino acid mature protein (Gharib et al., Endocrine Reviews, 11, 177 (1990)).
The gene encoding the beta subunit of TSH has also been characterized. The gene encoding TSH β-subunit has been cloned and sequenced in humans (Hayashizaki et al., FEBS Lett., 188, 363 (1985)), cattle (Maurer et al., J. Biol. Chem., 259, 5024 (1984)), mouse (Wolf et al., J. Biol. Chem., 262, 16596, (1987)), rat (Croyle et al., DNA, 5, 299 (1986)), and dog (Yang et al., Domestic Animal Endocrinology, 18, 363 (2000)). There are three exons and two introns in the β-subunit gene. The first is only 37 base pair (bp) and untranslated followed by a 3.9 kilo base intron. The function of the first exon is unclear; however it has been speculated that exon I may interact directly with thyroid hormone and its receptor and down regulate the TSHβ gene (Wondisford et al., J. Biol. Chem. 263, 12538 (1988)). However, while there has been extensive work on both thyrotropin subunits in a variety of different species, the DNA sequences and expressed glycoproteins for feline TSH have not yet been reported.
Feline TSH has been speculated to play an important part in the pathogenesis and diagnostic tests for feline hyperthyroidism, which has been recognized as the most common endocrine disorder in cats. One out of every three hundred cats is now diagnosed for hyperthyroidism. Canned cat food, cat litter, and pesticides have been identified as possible risk factors for the disease (Martin et al., J. Am. Vet. Med. Assoc., 217(6), 853 (2000)). The most common cause of hyperthyroidism is a thyroid adenoma that produces excessive circulating concentrations of triiodothyronine (T3) and thyroxine (T4). Feline hypertheyroidism occurs primarily in middle-aged to older cats, and can vary from mild to severe in effect. The most common symptoms associated with feline hyperthyroidism are weight loss, hyperactivity, increased appetite, and excessive drinking and urination. Intermittent vomiting and diarrhea may also occur, as well as increased heart rate and arrhythmias. Early diagnosis of feline hyperthyroidism is preferred, as untreated hyperthyroidism can lead to kidney disease, hypertension, and diabetes, and may eventually lead to congestive heart failure. Current diagnosis of feline hyperthyroidism is based on measurement of levels of T3 and T4, while current therapy utilizes antithyroid drug administration, surgery, and/or the use of radioactive iodine.
A commercially available canine TSH immunoassay (Williams et al., J. Am. Vet. Med. Assoc., 209, 1730 (1996)) has been evaluated for detection of feline TSH (fTSH). Although 68% of hyperthyroid cats had TSH concentrations below the detection limit, the assay was not sensitive enough to distinguish normal from low values (Graham et al., Proceedings, 18th ACVIM Forum, Seattle, Wash., Abstract p. 719 (2000)). Therefore, peptide reagents and antibodies that are more specific for feline thyrotropin are necessary for a clinically useful immunoassay. Measurement of endogenous fTSH would allow diagnosis of early hyperthyroidism where TSH levels are suppressed by a hyperfunctioning thyroid gland. Also, a valid feline TSH assay would help characterize chemicals that might directly or indirectly influence feline thyroid physiology, potentially leading to hyperthyroidism. The problem of obtaining sufficient thyrotropin for the development of such tests is complicated by the fact that a commercially available pituitary source of fTSH does not exist.